Indian Hedgehog,a Neglected Member of Hedgehog Pathway,May Offer a Novel Avenue for Colorectal Cancer Therapy

Introduction

One of the most intriguing recent insights into the genetic basis of cancer has been the realization that key pathways essential to the proper development of an embryo are frequently subverted in a wide range of tumors. ¹ Within this change of conceptual framework, cancer can no longer be framed purely in terms of a network of oncogenes and tumor-suppressor genes, thus giving novel insights into potential therapeutic strategy against cancer.

The hedgehog (Hh) signaling pathway plays a critical role in normal mammalian gastrointestinal development. ² In the past few years, ligand-dependent (i.e., ligand overexpression) activation of the Hh pathway has been shown in the formation and maintenance of a number of malignancies, including colorectal cancer (CRC). ^{3 –5} Although much work has been done to determine the exact role of this pathway in CRC over the past few years, the results of these studies were conflicting, ^{6 –9} and the effect of the Hh pathway inhibitor on the viability of colon cancer cells has been controversial. ^{7 –10}

There are three mammalian homologs of Hh ligands: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Briefly, when Hh ligands bind the transmembrane receptor, Patched1 (Ptch), Smoothened (Smo) suppression is relieved, leading to Gli entering the nucleus and activating transcription of Hh target genes. ¹¹ Of the three mammalian Hh homologs, Shh has been the most studied, mainly because of the paradigm that experiments with Shh protein are generally also applicable to other members of the family. ¹² However, recent studies challenge the functional redundancy of three Hh molecules by showing that Ihh is not a functional homolog of Shh activity during tumorigenesis. ^13,14

Ihh May Act as a Competitive Inhibitor of Shh

Some experimental assays indicated that three Hh ligands have similar biologic properties with differing potency (Shh>Ihh>Dhh), but others showed that each of the Hh homologs appears to have a unique set of roles, as suggested by their distinct expression domains and various functional studies. ^{15 –17} Recent studies have shown that, in the normal colon, Ihh protein is expressed in the superficial cells of the colon, while Shh protein is expressed at a very low level at the bases of the colonic crypt or not expressed at all. ¹⁸ Moreover, Ihh expression was reported to be lost from colonic adenomas. ¹⁴ In contrast, Shh was overexpressed in adenomas and adenocarcinomas of the colon. ^7,19 It seems likely that Shh and Ihh are expressed in a mutually exclusive pattern. This mutually exclusive distribution of Shh and Ihh in the colon is probably functionally important.

Indeed, recent reports have shown that exogenous Shh promotes cell proliferation in colonocytes, ^7,19 whereas Ihh regulates differentiation of mature colonocytes. ¹⁴ Further, previous analyses have indicated that the Ihh and Shh proteins have similar structure, ¹⁶ and Ihh is less active, in some assays, than Shh, ^13,16 thus possibly providing a basis for competitive inhibition of Shh. Consequently, Ihh may behave as a potent competitive inhibitor of Shh for binding to Ptch, and relative expressions of Ihh and Shh may play a role in maintaining the precise balance between proliferation and differentiation of the colonic epithelium. However, more direct evidence is needed to determine the relative contributions of Shh and Ihh in the homeostasis of the colonic epithelium.

Downregulation of Ihh Expression May Be an Important Step to the Establishment of the Malignant Colonic Phenotype

A previous report has shown that Ihh is expressed in the intervilli region of the small intestine. ²⁰ In Ihh^−/− mice, there is a significant reduction in size of the villi. ²⁰ Ihh has also been shown to be expressed in terminally differentiated absorptive colonocytes, and the colonic epithelium of Ihh^−/− embryos lost the ability to organize into crypts. ¹⁴ These results indicate a requirement of Ihh for colonic epithelial differentiation. When Hh signaling is blocked with cyclopamine, a specific inhibitor of Smo, the colonocytes at the luminal end of the crypt, show an enlarged nucleus and cytoplasm, with loss of Villin expression at the brush border and redistribution of Villin to the cytoplasm. ¹⁴ A lack of differentiation observed in colonic epithelial cells treated with Hh-signaling inhibitors mirrors that seen in colorectal carcinogenesis, suggesting that loss of Ihh may have a role in the development of dysplasia.

It is well known that activation of the Wnt pathway is one of the most important events involved in the multistep process of colorectal tumorigenesis. ²¹ Recently, a novel Wnt-Ihh axis in colonic epithelial renewal and carcinogenesis was proposed. ²² Loss of Ihh expression in dysplastic epithelial cells in colonic adenomas is seen in response to the activating of Wnt pathway. ¹⁴ Therefore, decreased expression of Ihh could be an important step to the establishment of the malignant colonic phenotype. Thus, Ihh may serve as a potential target for therapeutic intervention, although more work is needed to understand the exact role of Ihh in colon cancer progression.

Upregulation of Ihh Expression May Offer a Novel Therapeutic Approach Against CRC

Intervention of the Hh pathway has provided a therapeutic opportunity for treatment of malignancies. Effective inhibition of the Hh pathway can be achieved at the level of ligands by using anti-Hh antibodies, or through downstream effector molecules, such as Smo, with small-molecule antagonists. ²³ However, this raises concerns about the potential toxicity of systemic administration of Hh antagonists, which would block a critical developmental pathway. In addition, the Hh pathway inhibitor, cyclopamine, did not affect the viability of some colon cancer cells. ^9,22 Moreover, most methods currently available to manipulate Hh pathway activity are not specific for any Hh homolog, so there remains a degree of uncertainty. ²

Fortunately, recent advances toward understanding the role of Hh homologs raise hopes that greater specificity could possibly be achieved through upregulation of Ihh expression. A recent report has demonstrated that Ihh could repress Wnt signaling in colon cancer cells. ¹⁴ In addition, giving recombinant N-terminal Shh (91% identical to Ihh) leads to induction of Cip-1 and Villin expressions. ¹⁴

Conclusions

These data suggest that upregulation of Ihh may be necessary and sufficient for promoting the differentiation of colon cancer cells. Further, we hypothesize that upregulation of Ihh could competitively inhibit the binding of Shh to Ptch, while inhibition of Shh has been shown to decrease colonocytes proliferation in previous studies. ^7,19 Inspiringly, effective, specific, and safe therapy against CRC could be achieved with the upregulation of Ihh expression, thereby inducing differentiation of colorectal cancer cells and abrogating the Shh signaling that drives cancer cells' growth.